CN111449187B - Lactobacillus buchneri S-layer protein modified carvacrol/beta-cyclodextrin liposome and preparation method and antibacterial application thereof - Google Patents
Lactobacillus buchneri S-layer protein modified carvacrol/beta-cyclodextrin liposome and preparation method and antibacterial application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3481—Organic compounds containing oxygen
- A23L3/349—Organic compounds containing oxygen with singly-bound oxygen
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/70—Fixation, conservation, or encapsulation of flavouring agents
- A23L27/72—Encapsulation
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- A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
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- A23P10/00—Shaping or working of foodstuffs characterised by the products
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Abstract
The invention discloses a carvacrol/beta-cyclodextrin liposome modified by lactobacillus buchneri S-layer protein, a preparation method and an antibacterial application thereof, wherein the carvacrol/beta-cyclodextrin liposome is mainly prepared from the following raw materials in parts by weight by using a solvent acceptable for food: 1-5 parts of lactobacillus buchneri S-layer protein, 2-5 parts of carvacrol, 10-30 parts of beta-cyclodextrin, youqirl 100, 20-50 parts of soybean lecithin and 4-10 parts of cholesterol. According to the invention, the carvacrol is embedded by using the beta cyclodextrin, and then the carvacrol/beta-cyclodextrin inclusion compound is encapsulated in the liposome, so that the solubility of the carvacrol is further improved, the encapsulation rate of the carvacrol in the liposome is improved, the utilization rate of the carvacrol is improved, and the lactobacillus buchneri S-layer protein is modified to the surface of the liposome, so that the stability and the storage performance of the liposome can be improved, the lactobacillus buchneri S-layer protein and the carvacrol are synergistically bacteriostatic, the bacteriostatic effect of the liposome is remarkably improved, and the sustainable release effect on the production of pathogenic bacteria can be realized.
Description
Technical Field
The invention belongs to the technical field of carvacrol liposome, and particularly relates to an S-layer protein modified carvacrol/beta-cyclodextrin liposome, a preparation method and an antibacterial application thereof.
Background
Carvacrol (car) is a colorless to pale yellow liquid with thymol flavor, and is commonly found in essential oils of origanum vulgaris, thyme, guayule, and the like. At present, carvacrol is reported to have the effects of resisting oxidation, resisting bacteria, expelling parasites, relieving ischemia reperfusion injury of brain and spinal cord of rats and the like. Because the perfume has no toxic or side effect, no residue and no pollution to the environment, the perfume is often used as perfume to be added into daily necessities such as cosmetics and the like; the seasoning is often used as a natural seasoning spice, so that fishy smell in seafood, meat, fish and other foods can be removed, and the flavor of dishes is increased; is often used as a bacteriostatic agent and a preservative in food preservation. However, the carvacrol is insoluble in water, is easy to volatilize and oxidize in the air, has poor stability and the like, and greatly influences the bioavailability and the application range of the carvacrol.
In the prior art, CN201310436851 prepares carvacrol and beta-cyclodextrin into an inclusion compound to improve the stability of carvacrol. CN1618294 discloses an anti-mite composition with essential oil extracted from natural aromatic plant thyme as active component, and the 24-hour repellency rate reaches 100%. CN102178251A discloses a thyme mutton smell removing flavoring agent which has good smell removing effect, beautiful color, appropriate thyme fragrance, harmonious and natural taste, high application value and wide market prospect. CN104379156A discloses a thyme extract of thyme mullet for the treatment of inflammatory bowel disease. CN104478607A discloses a thymol bactericide and a preparation method of several dosage forms of the bactericide. The bactericide has low toxicity, environmental protection, safety and harmlessness, and is a bactericide with great market application prospect.
Although carvacrol is widely applied in the medical, food and cosmetic industries and has the characteristics of better sterilization, aroma enhancement and the like, carvacrol is volatile and unstable when exposed to air, so an effective method is sought for reducing the volatilization degree of carvacrol in the using process and prolonging the storage period of carvacrol. The beta-cyclodextrin is the only cyclodextrin product capable of being produced in large quantities in the industry at present due to simple production process and low cost. The beta cyclodextrin can wrap the carvacrol, enhance the solubility of the carvacrol and cannot damage the main active ingredients of the carvacrol. As described in the prior art CN201310436851, carvacrol and beta-cyclodextrin are prepared into an inclusion compound to improve the stability of carvacrol, and a related study that carvacrol is directly prepared into liposome also exists in the prior art, but the prior art also has the defects of low encapsulation efficiency and poor antibacterial performance and slow release performance of products.
The lactobacillus surface is covered with a layer of S-layer protein which is protein or glycoprotein with a symmetrical sub-lattice array structure formed by self-assembly and consists of single protein or glycoprotein subunits. The S-layer protein accounts for 10-15% of the total cell protein, and the relative molecular mass is between 40-200kDa. S-layer proteins can self-assemble on the surface of liposomes by electrostatic interaction in a suitable environment to form S-layer protein coated liposomes to use lipids for functionalization for various applications. Compared with common liposome, the S-layer protein coating liposome has excellent physicochemical and biological stability. S-layer proteins such as l.brevis and l.kefir can be used to encapsulate liposomes and maintain their stability in harsh environments such as high temperature, low pH, etc. And it was found that the affinity of the l.kefir glycosylated S-layer protein for liposomes was higher than that of the non-glycosylated l.brevis S-layer protein. But no study on S-layer protein modified liposomes on bacteriostasis was made.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the technical problems, the invention provides a carvacrol/beta-cyclodextrin liposome modified by lactobacillus buchneri S-layer protein, a preparation method and an antibacterial application thereof. According to the invention, the carvacrol is wrapped in the cyclodextrin liposome, so that the volatility of the carvacrol in the using process is reduced, the solubility of the carvacrol is enhanced, the utilization rate of the carvacrol is improved, the stability and the adhesion property of the carvacrol are enhanced by self-assembling the lactobacillus buchneri S-layer protein on the surface of the liposome, and the long-acting slow-release antibacterial effect is achieved.
The technical scheme is as follows: in order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
a lactobacillus buchneri S-layer protein modified carvacrol/beta-cyclodextrin liposome is mainly prepared from the following raw materials in parts by weight by utilizing a food-acceptable solvent:
1-5 parts of lactobacillus buchneri S-layer protein, 2-5 parts of carvacrol, 10-30 parts of beta-cyclodextrin, 20-50 parts of soybean lecithin, 20-50 parts of Youqirl and 4-10 parts of cholesterol.
Preferably, the food acceptable solvent is selected from the group consisting of absolute ethanol and ultra pure water.
Preferably, carvacrol and beta-cyclodextrin are firstly prepared into a carvacrol/beta-cyclodextrin inclusion compound, then soybean lecithin, youqirl 100 and cholesterol are utilized to load the carvacrol/beta-cyclodextrin inclusion compound into liposome, and finally S-layer protein is modified in the carvacrol/beta-cyclodextrin liposome to obtain the S-layer protein modified carvacrol/beta-cyclodextrin liposome.
The preparation method of the lactobacillus buchneri S-layer protein modified carvacrol/beta-cyclodextrin liposome comprises the following steps:
a. taking beta-cyclodextrin, carvacrol and ultrapure water as a solvent to prepare a beta-cyclodextrin inclusion compound of carvacrol, namely a carvacrol/beta-cyclodextrin inclusion compound;
b. re-dissolving the carvacrol/beta-cyclodextrin inclusion compound obtained in the step a in ultrapure water to prepare a carvacrol/beta-cyclodextrin inclusion compound aqueous solution, then preparing a soybean lecithin, youqirl 100 and cholesterol absolute ethyl alcohol mixed solution, injecting the mixed solution into the carvacrol/beta-cyclodextrin inclusion compound aqueous solution, and stirring;
c. and c, evaporating the solution prepared in the step b under reduced pressure to remove ethanol, and filtering by using a centrifugal and microporous filter membrane to obtain a carvacrol/beta-cyclodextrin liposome solution.
d. Dissolving the lactobacillus buchneri S-layer protein freeze-dried powder in carvacrol/beta-cyclodextrin liposome solution, and stirring to obtain the S-layer protein modified carvacrol/beta-cyclodextrin liposome.
Preferably, in the step a, the beta-cyclodextrin is added into ultrapure water for swelling to prepare a beta-cyclodextrin solution with the mass volume ratio of 0.01-99%, then carvacrol is added into the beta-cyclodextrin solution, and the carvacrol/beta-cyclodextrin inclusion compound is obtained after uniform mixing and freeze drying.
Preferably, in the step b, the soybean lecithin, the Ettky RL100 and the cholesterol are added into 50 to 100 parts of absolute ethyl alcohol to prepare a mixed solution, then the mixed solution is injected into the carvacrol/beta-cyclodextrin inclusion compound aqueous solution at the speed of 0.1 to 1mL/min, the volume ratio of the absolute ethyl alcohol mixed solution to the carvacrol/beta-cyclodextrin inclusion compound aqueous solution is 1: 0.1 to 2, and the stirring is carried out.
Preferably, in the step c, the reduced pressure evaporation temperature is 35-70 ℃, and the reduced pressure evaporation time is 20-40 min.
Preferably, in step d, the lactobacillus buchneri S-layer protein is a soluble protein extracted from lactobacillus buchneri, and is freeze-dried into powder, and further preferably, the molecular weight of the S-layer protein is 40-200kDa. The stirring is magnetic stirring for 1-4 h at 20-25 ℃.
Further, the extraction method of the lactobacillus buchneri S-layer protein comprises the following steps:
1) Culturing lactobacillus buchneri: the MRS solid culture medium and the MRS liquid culture medium are sequentially utilized to culture the lactobacillus buchneri, until the last logarithmic phase of the growth of the lactobacillus buchneri, three generations of strains are continuously inoculated to improve the activity of the strains, and the third generation of lactobacillus culture solution is reserved.
2) S-layer protein extraction: and (3) treating the cultured lactobacillus buchneri with a LiCl solution and a CuHCl solution in sequence, collecting supernatant, dialyzing, centrifuging, collecting the solution, and freeze-drying to obtain the lactobacillus buchneri S-layer protein.
The resulting S-layer protein was analyzed by SDS-PAGE, and the protein concentration of the solution was determined by BCA protein concentration determination kit three times each to obtain precision.
Preferably, in step 1), the lactobacillus buchneri strain is cultured in the MRS liquid medium for 18 to 23 hours.
Preferably, in the step 2), the concentration of the LiCl solution is 5-10 mol/mL, and the concentration of the CuHCl solution is 1-6mol/mL.
Wherein, the swelling time in the step a is preferably 1min to 48h.
In the step a, carvacrol can be uniformly mixed (i.e. completely dissolved) in the solvent by adopting a conventional method, for example, carvacrol is completely dissolved in the solvent by adopting methods such as standing, heating, stirring, ultrasonic or grinding and the like.
Wherein, the oxidation of the phospholipids can be caused by the over-high temperature of the reduced pressure evaporation in the step c, and the liposome can not be formed when the temperature is too low.
Wherein, the blank liposome is prepared by adding soybean lecithin, cholesterol and Ettky RL100 into 50-100 parts of absolute ethyl alcohol to prepare a mixed solution, then injecting the mixed solution into ultrapure water at the speed of 0.1-1 mL/min, stirring and decompressing and evaporating until the ethyl alcohol is completely volatilized.
The invention finally provides the application of the S-layer protein modified carvacrol/beta-cyclodextrin liposome as an antibacterial agent.
The invention firstly prepares carvacrol and beta-cyclodextrin into carvacrol/beta-cyclodextrin inclusion compound, then carries out loading on the beta-cyclodextrin/carvacrol inclusion compound through nano-liposome and self-assembles S-layer protein on the surface of the liposome, and finally prepares the S-layer protein modified carvacrol/beta-cyclodextrin liposome with nano-scale particle size. The nanometer liposome can reduce the volatility of carvacrol, prolong the shelf life and improve the liposome quality. Therefore, the beta-cyclodextrin/carvacrol inclusion compound is selected to prepare the liposome with higher stability, and the entrapment rate and the slow release performance of carvacrol can be further improved. In addition, due to the subcellular size of the nano-liposomes, the nano-liposomes can enhance the capability mechanism of the liposomes to contact with bacteria, so that the carvacrol essential oil has the antibacterial effect.
Has the beneficial effects that: compared with the prior art, the invention has the following advantages:
1. aiming at the problems that carvacrol is not easy to dissolve in water, is easy to volatilize and has low utilization rate, cyclodextrin liposome double-layer embedding is adopted, so that the dissolving problem of carvacrol is greatly increased, the utilization rate of carvacrol is improved, waste is reduced, and the cost is reduced to play an effective role.
2. In the food industry, carvacrol can be used as a spice and has broad-spectrum antibacterial property on common food-borne pathogenic bacteria. The novel carvacrol/beta-cyclodextrin liposome antibacterial agent is prepared by a cyclodextrin liposome delivery system, so that the biological activity of carvacrol is improved, the slow release characteristic is realized, and the storage life is prolonged.
3. The invention makes full use of the characteristics of the cyclodextrin liposome, not only improves the edible value of carvacrol, but also provides a new way for improving the added value of the antibacterial property of carvacrol.
4. The invention utilizes the S-layer protein of the lactobacillus buchneri to modify the carvacrol/beta-cyclodextrin liposome, can improve the stability and the adhesion property to bacteria, and leads the prepared liposome to have stronger stability.
Drawings
FIG. 1: and (3) extracting the lactobacillus buchneri S-layer protein.
FIG. 2: the entrapment rate of the S-layer protein modified carvacrol/beta cyclodextrin liposome is improved.
FIG. 3: the stability of carvacrol liposome and S-layer protein modified carvacrol/beta cyclodextrin liposome.
FIG. 4: carvacrol release curves in different liposomes.
Detailed Description
The technical solution of the present invention is further described in detail by the following specific examples.
The method for culturing lactobacillus buchneri in the following examples is as follows:
diluting Lactobacillus buchneri suspension to a certain gradient, coating on MRS solid culture medium, culturing in 37 deg.C constant temperature incubator for 48h, selecting single colony, inoculating to MRS liquid culture medium, standing in 37 deg.C constant temperature incubator for a certain time to late logarithm of growth of Lactobacillus buchneri, continuously inoculating third generation lactobacillus to improve the activity of strain, and storing the third generation lactobacillus culture solution in 4 deg.C refrigerator
Example 1
1) Extraction of lactobacillus buchneri S S-layer protein
1. Inoculating lactobacillus buchneri to MRS liquid culture medium, culturing for 20h in a 37 ℃ constant temperature incubator, centrifuging the bacterial liquid (8000 r/min,4 ℃ and 15 min), weighing thalli, adding 10mol/mL LiCl solution into each gram of thalli, acting for 0.5h in the 37 ℃ constant temperature incubator, centrifuging (8000 r/min,4 ℃ and 15 min), and then discarding the supernatant; adding 6mol/mL CuHCl solution into each gram, acting at 37 deg.C for 1h, centrifuging (8000 r/min,4 deg.C, 15 min), and collecting supernatant; transferring the supernatant into a dialysis bag with a molecular weight cut-off of 14kDa, dialyzing in deionized water at 4 ℃, and replacing water every 4 hours; dialyzing for 48h, centrifuging (12000 r/min,4 deg.C, 15 min) to collect solution as S-layer protein, freeze drying the obtained protein, storing in-20 deg.C refrigerator, and analyzing by SDS-PAGE, the result is shown in FIG. 1. The protein concentration of the solution was measured by the BCA protein concentration measurement kit three times each to obtain precision.
2) Preparation of carvacrol/beta cyclodextrin inclusion compound
1. Preparing a beta-cyclodextrin solution: weighing 12 parts of beta-cyclodextrin in 120 parts of ultrapure water, stirring and dissolving to prepare a beta-cyclodextrin solution for later use;
2. preparing a carvacrol/beta-cyclodextrin solution: adding 4 parts of carvacrol into the beta-cyclodextrin solution, magnetically stirring for 2h, centrifuging at 10000rpm/min for 10min, and filtering through a filter membrane of 0.22 mu m to obtain a filtrate, namely a carvacrol/beta-cyclodextrin inclusion compound aqueous solution;
3) Preparation of S-layer protein modified carvacrol/beta cyclodextrin liposome
Weighing 20 parts of soybean lecithin, 100 parts of Ettky RL and 20 parts of cholesterol. Adding 100 parts of absolute ethyl alcohol solution for dissolving, uniformly mixing to prepare a mixed solution, then injecting the mixed solution into carvacrol/beta cyclodextrin inclusion compound aqueous solution or ultrapure water at the speed of 0.5mL/min, wherein the volume ratio of the absolute ethyl alcohol mixed solution to the carvacrol/beta cyclodextrin inclusion compound aqueous solution is 1: 2, magnetically stirring for 15min, and rotationally evaporating to remove the absolute ethyl alcohol until the solution is 100 parts to form carvacrol/beta cyclodextrin liposome or blank liposome. The reduced pressure evaporation temperature is 50 deg.C, and the reduced pressure evaporation time is 30min. Centrifuging the obtained carvacrol/beta cyclodextrin liposome liquid, and filtering with a sterile filter membrane of 0.22 mu m to obtain filtrate. Dissolving the S-layer protein freeze-dried powder in carvacrol/beta-cyclodextrin liposome, magnetically stirring for 2h at 22 ℃ to obtain the S-layer protein modified carvacrol/beta-cyclodextrin liposome, and storing at 4 ℃ for later use.
Example 2
1) Extraction of lactobacillus buchneri S S-layer protein
1. Inoculating lactobacillus buchneri to an MRS liquid culture medium, culturing for 21h in a constant-temperature incubator at 37 ℃, centrifuging the bacterial liquid (8000 r/min,4 ℃ and 15 min), weighing thalli, adding 8mol/mL LiCl solution into each gram of thalli, acting for 0.5h in the constant-temperature incubator at 37 ℃, centrifuging (8000 r/min,4 ℃ and 15 min), and then discarding the supernatant; adding 5mol/mL CuHCl solution into each gram, acting at 37 deg.C for 1h, centrifuging (8000 r/min,4 deg.C, 15 min), and collecting supernatant; transferring the supernatant into a dialysis bag with a molecular weight cut-off of 14kDa, dialyzing in deionized water at 4 ℃, and replacing water every 4 hours; dialyzing for 48h, centrifuging (12000 r/min,4 deg.C, 15 min) to collect solution as S-layer protein, freeze drying the obtained protein, storing in-20 deg.C refrigerator, and analyzing by SDS-PAGE. The protein concentration of the solution was measured by the BCA protein concentration measurement kit three times each to obtain precision.
2) Preparation of carvacrol/beta cyclodextrin inclusion compound
1. Preparing a beta-cyclodextrin solution: weighing 10 parts of beta-cyclodextrin in 100 parts of ultrapure water, stirring and dissolving to prepare a beta-cyclodextrin solution for later use;
2. preparing a carvacrol/beta-cyclodextrin solution: adding 3 parts of carvacrol into the beta-cyclodextrin solution, magnetically stirring for 2h, centrifuging at 10000rpm/min for 10min, and filtering through a filter membrane of 0.22 mu m to obtain a filtrate, namely a carvacrol/beta-cyclodextrin inclusion compound aqueous solution;
3) Preparation of S-layer protein modified carvacrol/beta cyclodextrin liposome
Weighing 15 parts of soybean lecithin, 15 parts of Ettky RL and 8 parts of cholesterol. Adding 80 parts of absolute ethyl alcohol solution for dissolving, uniformly mixing to prepare a mixed solution, then injecting the mixed solution into carvacrol/beta cyclodextrin inclusion compound aqueous solution or ultrapure water at the speed of 0.08mL/min, wherein the volume ratio of the absolute ethyl alcohol mixed solution to the carvacrol/beta cyclodextrin inclusion compound aqueous solution is 1: 0.1, magnetically stirring for 15-60min, and then rotationally evaporating to remove the absolute ethyl alcohol until the total volume of the solution is 20mL to form carvacrol/beta cyclodextrin liposome or blank liposome. The reduced pressure evaporation temperature is 40 deg.C, and the reduced pressure evaporation time is 40min. Centrifuging the obtained carvacrol/beta cyclodextrin liposome liquid, and filtering with a sterile filter membrane of 0.22 mu m to obtain filtrate. Dissolving the S-layer protein freeze-dried powder in carvacrol/beta-cyclodextrin liposome, magnetically stirring for 3 hours at 22 ℃ to obtain the carvacrol/beta-cyclodextrin liposome modified by the S-layer protein, and storing at 4 ℃ for later use.
Example 3
1) Extraction of lactobacillus buchneri S S-layer protein
1. Inoculating lactobacillus buchneri to MRS liquid culture medium, culturing for 22h in a 37 ℃ constant temperature incubator, centrifuging the bacterial liquid (8000 r/min,4 ℃ and 15 min), weighing thalli, adding 6mol/mL LiCl solution into each gram of thalli, acting for 0.5h in the 37 ℃ constant temperature incubator, centrifuging (8000 r/min,4 ℃ and 15 min), and then discarding the supernatant; adding 3mol/mL CuHCl solution into each gram, acting at 37 deg.C for 1h, centrifuging (8000 r/min,4 deg.C, 15 min), and collecting supernatant; transferring the supernatant into a dialysis bag with a molecular weight cut-off of 14kDa, dialyzing in deionized water at 4 ℃, and changing water every 4 h; dialyzing for 48h, centrifuging (12000 r/min,4 deg.C, 15 min) to collect solution as S-layer protein, freeze drying the obtained protein, storing in-20 deg.C refrigerator, and analyzing by SDS-PAGE. The protein concentration of the solution was measured by the BCA protein concentration measurement kit three times each to obtain precision.
2) Preparation of carvacrol/beta cyclodextrin inclusion compound
1. Preparing a beta-cyclodextrin solution: weighing 10 parts of beta-cyclodextrin in 100 parts of ultrapure water, stirring and dissolving to prepare a beta-cyclodextrin solution for later use;
2. preparing a carvacrol/beta-cyclodextrin solution: adding 3 parts of carvacrol into the beta-cyclodextrin solution, magnetically stirring for 2h, centrifuging at 10000rpm/min for 10min, and filtering through a filter membrane of 0.22 mu m to obtain a filtrate, namely a carvacrol/beta-cyclodextrin inclusion compound aqueous solution;
2) Preparation of S-layer protein modified carvacrol/beta cyclodextrin liposome
Weighing 10 parts of soybean lecithin, 10 parts of Ettky RL and 5 parts of cholesterol. Adding 50 parts of absolute ethyl alcohol solution for dissolving, uniformly mixing to prepare a mixed solution, then injecting the mixed solution into carvacrol/beta cyclodextrin inclusion compound aqueous solution or ultrapure water at the speed of 0.05mL/min, wherein the volume ratio of the absolute ethyl alcohol mixed solution to the carvacrol/beta cyclodextrin inclusion compound aqueous solution is 1: 1, magnetically stirring for 15-60min, and performing rotary evaporation to remove the absolute ethyl alcohol until the total volume of the solution is 20mL to form carvacrol/beta cyclodextrin liposome or blank liposome. The reduced pressure evaporation temperature is 60 deg.C, and the reduced pressure evaporation time is 20min. Centrifuging the obtained carvacrol/beta cyclodextrin liposome liquid, and filtering with a sterile filter membrane of 0.22 mu m to obtain filtrate. Dissolving the S-layer protein freeze-dried powder in carvacrol/beta-cyclodextrin liposome, magnetically stirring for 1.5 hours at 22 ℃ to obtain the S-layer protein modified carvacrol/beta-cyclodextrin liposome, and storing at 4 ℃ for later use.
Preparation of carvacrol liposomes
Weighing 10 parts of soybean lecithin, 100 parts of Uttky RL, 5 parts of cholesterol and 1 part of carvacrol, adding 50 parts of absolute ethyl alcohol solution for dissolving, uniformly mixing, injecting into ultrapure water to prepare a mixed solution, wherein the volume ratio of absolute ethyl alcohol to the ultrapure water solution is 1: 2, magnetically stirring for 15min, and performing rotary evaporation to remove the absolute ethyl alcohol until the total volume of the solution is 20ml to form carvacrol liposome. Filtering the obtained carvacrol liposome liquid with 0.22 μm sterile filter membrane to obtain filtrate. Storing at 4 deg.C for use.
Examples of the experiments
Samples from example 1 and the control were taken for the following tests:
1. determination of encapsulation efficiency
1. Diluting carvacrol with absolute ethyl alcohol, accurately weighing a certain proportion of carvacrol standard substance, placing the carvacrol standard substance into a 250mL volumetric flask, adding a proper amount of absolute ethyl alcohol solution to dissolve the carvacrol standard substance, metering volume to a scale mark, and shaking up. Then, precisely absorbing a certain volume of the above solutions to prepare 0.005-0.1mg/mL carvacrol ethanol standard solution, measuring absorbance values at 275nm (each concentration is repeatedly measured for 3 times, and the result is averaged), and obtaining a standard curve linear regression equation of y =11.219x +0.0152 (R =11.219x +) 2 = 0.9997), wherein x is the carvacrol concentration (mg/mL), y is the absorbance value, and the linear concentration range is 0.061 mg/mL-1.128 mg/mL. 1mL of S-layer protein modified carvacrol/beta cyclodextrin liposome sample was centrifuged at 10000rpm for 4 minutes, the supernatant was removed and the collected precipitate was redissolved in an equal volume of ethanol solution by sonication for 1 hour. Finally, the mixture was centrifuged at 6000rpm at 4 ℃ for 10 minutes and passed through an ultraviolet spectrophotometer methodThe supernatant was collected by analysis. The encapsulation efficiency of carvacrol in liposomes was calculated by the following equation:
EE(％)＝C 1 /C 0 ×100％，
wherein, EE is the encapsulation rate (%),
C 1 the concentration (mg/mL) of carvacrol in the S-layer protein modified carvacrol/beta cyclodextrin liposome is adopted,
C 0 is the initial concentration of carvacrol (mg/mL).
2. Entrapment rate of S-layer protein modified carvacrol/beta cyclodextrin liposome
The entrapment rate is the most important index for evaluating the quality of the liposome preparation, and is also the key for judging whether the liposome can exert the characteristics of high efficiency, low toxicity and the like compared with the common preparation. As can be seen from figure 2, the entrapment rate of the carvacrol liposome is 14.3%, and the entrapment rate of the S-layer protein modified carvacrol/beta cyclodextrin liposome is 73%. Therefore, the prepared S-layer protein modified carvacrol/beta cyclodextrin liposome can obviously improve the entrapment rate of the liposome.
2. Protein coating rate of S-layer protein modified carvacrol/beta cyclodextrin protein liposome
The coating rate of the S-layer protein, namely the content of the S-layer protein self-assembled on a liposome membrane, is indirectly quantified by adopting a BCA method trace protein detection kit, and after the S-layer protein modified carvacrol/beta-cyclodextrin liposome is centrifuged at 15000rpm/min for 30min, the content of the free protein in the supernatant is determined. The calculation formula of the protein coating rate is as follows:
P＝(1-C 1 /C 0 )×100％
wherein P is a protein coating rate (%),
C 0 initial concentration of S-layer protein (mg/mL),
C 1 is the concentration of free S-layer protein (mg/mL).
The protein coating rate is an important index for evaluating the quality of the proteoliposome preparation, and is also a key for judging whether the liposome can exert the characteristics of high efficiency, low toxicity and the like compared with the common preparation. As a result, the adsorption rate of S-layer protein was found to be 75.61%.
3. Antibacterial property of S-layer protein modified carvacrol/beta cyclodextrin liposome
1. Experimental Material
(1) Carvacrol
(2) Carvacrol liposome
(3) S-layer protein modified carvacrol/beta cyclodextrin liposome (beta-CD/Car/SLP-LP)
2. Experimental methods
The Minimum Inhibitory Concentration (MIC) of carvacrol, carvacrol liposome and S-layer protein modified carvacrol/beta cyclodextrin liposome is determined by adopting a trace broth dilution method, namely, 100 mu L of LB liquid culture medium is added into each hole of a 96-hole plate, then 100 mu L of bacteriostatic agent is added into each row of first holes, the first holes are fully blown and uniformly mixed by a gun head, 100 mu L of bacteriostatic agent is absorbed into the 2 nd hole, the first holes are sequentially diluted into the 11 th hole of the same row in a two-time series manner, and 100 mu L of bacteriostatic agent is absorbed and discarded after the 11 th hole is uniformly mixed. Then 100 mul of staphylococcus aureus bacterial liquid (10 mu L) diluted by normal saline is added into 1-10 holes and 12 th hole of each row 7 CFU/mL), 100. Mu.L of physiological saline was added to well 11. The plate was placed in a 37 ℃ incubator for 24 hours of static culture. The results were considered valid when all negative control wells (12 wells) were turbid and all positive control wells (11 wells) were clear. Determination of OD in each well Using microplate reader 600nm And (3) obtaining the MIC of carvacrol, carvacrol liposome and S-layer protein modified carvacrol/beta cyclodextrin liposome by using the negative hole with the lowest bacteriostatic concentration. Thereby evaluating the antibacterial activity of the carvacrol liposome and the carvacrol/beta cyclodextrin liposome modified by the S-layer protein. The experiment was repeated three times and the results averaged.
3. Antibacterial property of S-layer protein modified carvacrol/beta cyclodextrin liposome
The change of the antibacterial activity of carvacrol samples subjected to different treatments can also directly reflect that the antibacterial ability of the carvacrol samples can be improved by the cyclodextrin liposome embedding. Therefore, the minimum inhibitory concentration of carvacrol, carvacrol liposome and S-layer protein modified carvacrol/beta cyclodextrin liposome is determined, the results are shown in Table 1, 1-11 are respectively inhibitory solutions diluted by times, 12 is a blank control, the carvacrol solution only has No. 1 hole without bacteria, and the MIC value of carvacrol in the hole is 320 mu g/mL. Carvacrol liposomes were clarified in well 2, where the carvacrol MIC value was 160 μ g/mL. The beta-CD-carvacrol liposome modified by the S-layer protein is clarified in a No. 4 hole, and the MIC value is proved to be 40 mu g/mL. The S-layer protein modified carvacrol/beta cyclodextrin liposome has the advantages that the antibacterial effect of staphylococcus aureus is enhanced, the using amount is reduced, and the same antibacterial effect is achieved.
Table 1 antimicrobial susceptibility of s.aureus to S-layer protein coated beta-CD-carvacrol liposomes as measured by Minimum Inhibitory Concentration (MIC)
4. Stability of S-layer protein modified carvacrol/beta cyclodextrin liposome
1. Experimental Material
(1) Carvacrol/beta cyclodextrin liposome
(2) S-layer protein modified carvacrol/beta cyclodextrin liposome
2. Experimental methods
Membrane stability by measuring the detergent resistance of S-layer protein modified carvacrol/beta cyclodextrin liposomes. Briefly, the liposomes of the treatment group were observed by pipetting 100. Mu.L of 1% Triton X-100 into the liposome solution, mixing and shaking, adding 10. Mu.L of 1% Triton X-100 vortexing, and centrifuging at 8000rpm for 5 minutes before and after treatment, respectively, the two liposomes were added to the centrifuge tube.
The results are shown in fig. 3, wherein (a), (B), (C), and (D) are all β -CD-carvacrol liposomes coated with S-layer proteins at different concentrations (30, 40, 50, 100 μ g/mL surface layer protein); and (E) is beta-CD-carvacrol liposome. The upper graph was obtained by centrifugation at 8000rpm for 5 minutes after addition of 10. Mu.L 1% Triton100 vortexing, and it was found that the liposomes that were not modified with S-layer proteins precipitated the most, indicating that the liposome membranes were unstable and the most damaged. After the modification of S-layer protein, almost no precipitation is generated in the A (30 mu g/mL) and B (40 mu g/mL) liposomes, which represents that the liposome membrane stability is remarkably improved. The increased C and D precipitates are probably due to the increased amount of surface proteins and thus to the increased precipitates. Modification of S-layer protein reduces membrane fluidity, thereby hindering lipid extraction of high density lipoprotein, reducing risk of liposome decomposition, and thus improving its stability.
5. Slow release performance of S-layer protein modified carvacrol/beta cyclodextrin liposome
1. Experimental Material
(1) Carvacrol liposome
(2) S-layer protein modified carvacrol/beta cyclodextrin liposome
2. Experimental methods
Dynamic membrane dialysis method is adopted to carry out microsphere in vitro release test. Precisely weighing 6 parts of carvacrol/beta cyclodextrin liposome powder, wherein each part is about 3mg, adding 2mL of release medium into the carvacrol/beta cyclodextrin liposome powder for suspension, placing the carvacrol/beta cyclodextrin liposome powder into a pretreated dialysis bag, fastening the bag opening, placing the carvacrol/beta cyclodextrin liposome powder into 28mL of release medium, placing a sample into a constant-temperature magnetic stirrer, controlling the temperature at 37 ℃, controlling the rotating speed at 100rpm, periodically sucking the dialyzate and replenishing equivalent amount in time, filtering, measuring the concentration of carvacrol after dilution, and calculating the cumulative release percentage according to the same formula. The operation was repeated for each batch of samples. The mean values are plotted on the ordinate and the release times on the abscissa to plot the in vitro release curve.
The stability of the cyclodextrin liposome can be characterized by determining the slow release capability of the carvacrol liposome and the S-layer protein modified carvacrol/beta cyclodextrin liposome. As shown in fig. 4, carvacrol/cyclodextrin liposomes modified by S-layer protein are significantly better in sustained release performance than carvacrol directly embedded in liposomes.
Claims (5)
1. A preparation method of lactobacillus buchneri S-layer protein modified carvacrol/beta-cyclodextrin liposome is characterized by mainly comprising the following raw materials in parts by weight and utilizing a solvent acceptable for food:
1 to 5 parts of lactobacillus buchneri S-layer protein, 2 to 5 parts of carvacrol, 10 to 30 parts of beta-cyclodextrin, 20 to 50 parts of soybean lecithin, 20 to 50 parts of Youqirl and 4 to 10 parts of cholesterol, and the method comprises the following steps:
a. taking beta-cyclodextrin, carvacrol and ultrapure water as solvents to prepare a beta-cyclodextrin inclusion compound of the carvacrol, namely a carvacrol/beta-cyclodextrin inclusion compound;
b. re-dissolving the carvacrol/beta-cyclodextrin inclusion compound obtained in the step a in ultrapure water to prepare a carvacrol/beta-cyclodextrin inclusion compound aqueous solution, then preparing a soybean lecithin, equisiterl 100 and cholesterol absolute ethyl alcohol mixed solution, namely adding the soybean lecithin, the Equisiterl 100 and the cholesterol into 50 to 100 parts of absolute ethyl alcohol to prepare a mixed solution, then injecting the mixed solution into the carvacrol/beta-cyclodextrin inclusion compound aqueous solution at the speed of 0.1 to 1mL/min, wherein the volume ratio of the absolute ethyl alcohol mixed solution to the carvacrol/beta-cyclodextrin inclusion compound aqueous solution is 1: 0.1-2, and stirring;
c. evaporating the solution prepared in the step b under reduced pressure to remove ethanol, and filtering the solution through a centrifugal and microporous filter membrane to obtain a carvacrol/beta-cyclodextrin liposome solution, wherein the reduced pressure evaporation temperature is 35 to 70 ℃, and the reduced pressure evaporation time is 20 to 40min;
d. dissolving the lactobacillus buchneri S-layer protein freeze-dried powder in carvacrol/beta-cyclodextrin liposome solution, and stirring to obtain the S-layer protein modified carvacrol/beta-cyclodextrin liposome.
2. The preparation method of the lactobacillus buchneri S-layer protein modified carvacrol/beta-cyclodextrin liposome of claim 1, wherein in the step a, the beta-cyclodextrin is firstly added into ultrapure water to swell, so as to prepare a beta-cyclodextrin solution with the mass volume ratio of 0.01-99%, then the carvacrol is added into the beta-cyclodextrin solution, and the carvacrol/beta-cyclodextrin inclusion compound is obtained after the carvacrol is uniformly mixed and freeze-dried.
3. The method for preparing lactobacillus buchneri S-layer protein-modified carvacrol/beta-cyclodextrin liposome according to claim 1, wherein in step d, lactobacillus buchneri S-layer protein is soluble protein extracted from lactobacillus buchneri, and is freeze-dried into powder; the stirring is carried out at 20-25 ℃ for 1-4h by magnetic stirring.
4. The lactobacillus buchneri S-layer protein modified carvacrol/beta-cyclodextrin liposome obtained by the preparation method of any one of claims 1 to 3.
5. Use of the lactobacillus buchneri S-layer protein modified carvacrol/β -cyclodextrin liposomes of claim 4 for the preparation of an antibacterial agent.
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| CN106659683A (en) * | 2014-01-14 | 2017-05-10 | 约翰斯·霍普金斯大学 | Liposome compositions encapsulating modified cyclodextrin complexes and uses thereof |
| CN105997561B (en) * | 2016-07-04 | 2019-04-02 | 江苏大学 | A kind of preparation method and purposes of casein-thyme essential oil liposome antibacterial agent |
| CN106822918A (en) * | 2017-02-15 | 2017-06-13 | 南京师范大学 | A kind of surface modification liposome and its preparation method and application |
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